Voltage-gated sodium channel NaV1.5 is responsible for initiating and propagating cardiac action potentials by selectively conducting Na+ into cardiomyocytes. Class-I antiarrhythmic drugs target NaV1.5 for treatment of arrhythmias. During the last few years, cryogenic electron microscopy (cryo-EM) has become a powerful technique to determine the structures of ion channels at atomic level. In order to reveal the structural features of NaV1.5 and the structural basis for its interaction with antiarrhythmic drugs by cryo-EM, NaV1.5 protein must be expressed at high levels and purified to homogeneity. In this chapter, we discuss the expression and purification of NaV1.5 in a mammalian expression system. We optimized the construct by deleting unstructured intracellular loops of rat NaV1.5 while retaining core functional regions. The resulting rNaV1.5C is fully functional and is blocked by Class-I antiarrhythmic drugs in a state-dependent manner. Protocols are presented for expressing and purifying sufficient sample of NaV1.5 for preparing cryo-EM grids. The resulting cryo-EM structure is briefly described.The transient receptor potential ankyrin 1 (TRPA1) ion channel is a member of the TRP channel family that is involved in sensing noxious stimuli that elicit pain and inflammation. Because of its critical physiological role and therapeutic importance, great efforts have been made to understand the structure and mechanism of TRPA1. Several human TRPA1 structures have been reported using single particle cryo-electron microscopy (cryo-EM) over the last 6 years. Here, we present a protocol for the heterologous expression, large-scale purification, and nanodisc reconstitution of the human TRPA1 channel for cryo-EM and biochemical studies.The transient receptor potential (TRP) vanilloid 2 (TRPV2) and TRP vanilloid 5 (TRPV5) cation channels play an important role in various physiological and pathophysiological processes. The heterologous expression and purification of these channels is critical for functional and structural characterization of these important proteins. Full-length rat TRPV2 and rabbit TRPV5 can both be expressed in Saccharomyces cerevisiae and affinity purified using the 1D4 epitope and antibody to yield pure, functional channels. Further, these channels can be reconstituted into lipid nanodiscs for a more functionally relevant environment. Presented here are protocols for the expression of full-length rat TRPV2 and rabbit TRPV5 in Saccharomyces cerevisiae, their affinity purification, and their reconstitution into nanodiscs for structural and functional studies.Most membrane proteins, and ion channels in particular, assemble to multimeric biological complexes. This starts with the quarternary structure and continues with the recruitment of auxiliary subunits and oligomerization or clustering of the complexes. While the quarternary structure is best determined by atomic-scale structures, stoichiometry of heteromers and dynamic changes in the assembly cannot necessarily be investigated with structural methods. Here, single subunit counting has proven a powerful method to study the composition of these complexes. Single subunit counting uses the irreversible photodestruction of fluorescent tags as means to directly count a labeled subunit and thereby derive the composition of the assemblies. In this chapter, we discuss single subunit counting and its limitations. We present alternative methods and provide a detailed protocol for recording and analysis of single subunit counting data.The modulation of ion channel activity is of central importance within the nervous system, and an in-depth understanding of how such activity occurs on the molecular level is of prime importance for enhancing our understanding of neuronal systems in physiological and pathological states. https://www.selleckchem.com/products/valemetostat-ds-3201.html The use of light as a stimulus has presented the unique opportunity to study these dynamic processes with exquisite spatiotemporal control. We have developed the photoswitchable tweezers method, an optogenetic pharmacology-based technique which relies on the use of a photoswitchable crosslinker as "tweezers" to manipulate the molecular movements involved in ion channel functionalities. Not only does this allow optical control of ion channel activity, but also investigation into the molecular motions and inter-residue distances implicated in such activity. In this chapter we discuss the principles behind the photoswitchable tweezers method, its strategic design and the key experimental steps involved in this technique, using purinergic P2X2 receptor as a case study system.Ion channels are macromolecular complexes whose functions are exquisitely tuned by interacting proteins. Fluorescence resonance energy transfer (FRET) is a powerful methodology that is adept at quantifying ion channel protein-protein interactions in living cells. For FRET experiments, the interacting partners are tagged with appropriate donor and acceptor fluorescent proteins. If the fluorescently-labeled molecules are in close proximity, then photoexcitation of the donor results in non-radiative energy transfer to the acceptor, and subsequent fluorescence emission of the acceptor. The stoichiometry of ion channel interactions and their relative binding affinities can be deduced by quantifying both the FRET efficiency and the total number of donors and acceptors in a given cell. In this chapter, we discuss general considerations for FRET analysis of biological interactions, various strategies for estimating FRET efficiencies, and detailed protocols for construction of binding curves and determination of stoichiometry. We focus on implementation of FRET assays using a flow cytometer given its amenability for high-throughput data acquisition, enhanced accessibility, and robust analysis. This versatile methodology permits mechanistic dissection of dynamic changes in ion channel interactions.Despite major advances in methodologies for membrane protein production over the last two decades, there remain challenging protein complexes that are technically difficult to yield by conventional recombinant expression methods. A large number of these proteins are multimeric membrane proteins from eukaryotic species, which are required to pass through stringent quality control mechanisms of host cells for proper folding and complex assembly. Here, we describe the development procedure to improve the production efficiency of multi-oligomeric membrane protein complexes in insect cells and recombinant baculovirus, which involves screening of promoters, enhancers, and untranslated regions for expression levels, using calcium homeostasis modulator (CALHM) and N-methyl-d-aspartate receptor (NMDAR) proteins as examples. We demonstrate that our insect cell expression strategy is effective in expression of both multi-homomeric CALHM proteins and multi-heteromeric NMDARs.